Organic dicarboxylic acids, salts and preparation method thereof

ABSTRACT

Organic dicarboxylic acid compounds, salts and preparation methods thereof. The said compounds have activity of resisting oxidation damage to crystalline lens of eyes. The structures of the above organic dicarboxylic acid compounds are shown as formula (1).

FIELD OF THE INVENTION

The present invention relates to organic dicarboxylic acids, salts andthe preparation method thereof, as well as the activity of resistingoxidative damage to crystalline lens of some typical compounds in thesaid salts.

BACKGROUND OF THE INVENTION

Cataract is one of the most common eye diseases which lead to blindness.The blindness caused by cataract accounts for about 40% of the blindpeople all over the world. With the coming of the aging society, theincidence of senile cataract gradually increases, thus cataract hasbecome a worldwide common and frequently occurring disease. Thoughcrystalline lens deprived of vision can be removed and replaced withartificial crystalline lens by operations, the cost for such operationsis high and certain risks may be encountered. Therefore, more patientsrequire drug therapy to postpone the progression of crystalline lensopacification at the initial stage in order to avoid the loss of vision,postpone or avoid operations. It has been known that bendazac lysine hascertain effects in resisting cataract and it also has certaintherapeutic efficacy on polysaccharide cataract when used as aldosereductase inhibitor, but this drug has irritation on eyes and itstherapeutic efficacy is still insufficient. Therefore, it is essentialto develop a new generation of drugs to treat cataract.

CONTENTS OF THE INVENTION

Purpose of the invention: the present invention provides organicdicarboxylic acids, salts and preparation method thereof, wherein saidcompounds can be used as drugs for treating cataract.

Technical Solution: the present invention designs and synthesizes2-(1-benzyl (or substituent benzyl)-1H-indazole-3-oxyl) malonic acid,which is used as organic diacid to react with base metal ions, ammoniumion or amino acids to prepare a series of organic malonates with novelstructures. The purpose is to meet the required low-toxicity,low-irritation, excellent water solubility and effective activity ofantioxidation of crystalline lens in order to treat human cataract. Thepresent invention discloses a group of dicarboxylic acids and its saltswith novel structures. Using 2-(1-benzyl (or substituentbenzyl)-1H-indazole-3-oxyl) malonic acid as the organic dicarboxylicacid, the structure of these compounds can be shown by formula (1),

wherein, R is hydrogen atom, C₁₋₃ alkyl group, C₁₋₃ alkoxyl or halogenatom, and the compound shown in formula (1) is abbreviated as H₂L.

When the organic dioic acid shown in formula (1) reacts with the cationM to form salt, the salt can be represented by formula (2),

M₂L   formula (2)

wherein, M stands for monovalent cation, such as Na⁺, K⁺, NH₄ ⁺, etc.

When the the organic dioic acid shown in formula (1) reacts with aminoacids to form salt, the salt can be represented by formula (3),

A₂H₂L   formula (3)

wherein, A stands for amino acids, such as lysine (including L-lysine,D-lysine, and the raceme composed of L-lysine and D-lysine), histidine(including L-histidine, D-histidine, and the raceme composed ofL-histidine and D-histidine), etc.

Another purpose of the present invention is to provide the method forpreparing the organic dicarboxylic acid shown in formula (1), which canbe obtained from the reaction shown in formula (4),

wherein, X stands for Cl, Br or I atom, the definition for R is the sameas that in formula (1), and R′ stands for C₁₋₄ alkyl group. In thesynthesis for these organic diacids, 1-benzyl (or substituentbenzyl)-1H-indazole-3-alcohol (abbreviated as S1) reacts withα-halogenated malonic diester at the presence of alkali to obtain2-(1-benzyl (or substituent benzyl)-1H-indazole-3-oxyl) malonic diester(abbreviated as S2). Afterwards, the ester is hydrolyzed under alkalineconditions, and then after acidification the corresponding organicdiacid (H₂L) is obtained.

The detailed synthetic process is as followed: 1.0 mole of S1 issubjected to reflux reaction with 1.2 mole of XCH(CO₂R′)₂ at thepresence of 2.5 moles of potassium carbonate in the solvent of glycoldimethyl ether to obtain S2, subsequently S2 is subjected to refluxhydrolysis in potassium hydroxide (or sodium hydroxide) solution, andthen acidified to pH of about 2 with diluted hydrochloric acid, finallyH₂L is obtained.

The organic diacid (H₂L) shown in formula (1) is subjected to reactionwith corresponding alkali, such as sodium hydroxide, potassiumhydroxide, aqueous ammonia, etc., to obtain the salt (M₂L) shown informula (2); the organic diacid (H₂L) shown in formula (1) is subjectedto reaction with corresponding amino acid, such as lysine (includingL-lysine, D-lysine, and the raceme composed of L-lysine and D-lysine),histidine (including L-histidine, D-histidine, and the raceme composedof L-histidine and D-histidine) or other amino acids, to produce thesalt (A₂H₂L) as shown in formula (3).

The structures of the prepared compounds in the present invention havebeen confirmed by different analytical methods such as infrared spectra,proton nuclear magnetic resonance spectra, organic mass spectrometry orelectrospray ionization mass spectrometry.

Beneficial Effects: the present invention provides a kind of organicdicarboxylic acid, salt and preparation method thereof, and it disclosesthe activity of resisting oxidative damage to crystalline lens of sometypical organic dicarboxylate compounds, the irritation to the eyes ofwhich is lower than those of existing drugs, and the therapeuticefficacy of which is better.

Embodiments

The present invention is further illustrated by the followingembodiments, but this invention is not restricted to these embidiments.EXAMPLE 1

Preparation of 2-(1-benzyl(or substituent benzyl)-1H-indazole-3-oxyl)malonic ester 12 g (0.090 mol) 3-hydroxyl-indazole (J & K Scientific),3.94 g (0.098 mol) sodium hydroxide and 90 mL water is added into a 250mL three-necked bottle and agitated at 40° C. for 10 min, then 0.090 molbenzyl chloride (or substituent benzyl chloride) is dropwise added inand the mixture is reacted at 70° C. for 2h with solids beingprecipitated out, filter, the filter cake is washed with water andfinally 1-benzyl(or substituent benzyl)-1H-indazole-3-alcohol isobtained.

40 mL glycol dimethyl ether (solvent) is added into a 100 mL four-neckedbottle, then 1-benzyl(or substituenti benzyl)-1H-indazole-3-alcohol(10.5 mmol) and 3.6 g (26.1 mmol) potassium carbonate are mixed in thefour-necked bottle. The mixture is agitated at room temperature for 10min, subsequently 3.0 g (12.6 mmol) brominated diethyl malonate (J & KScientific) is dropwise added, afterwards the mixture is heated andsubjected to reflux for 4h, filter after the solution changes fromyellow into reddish brown. The filtrate is concentrated and subjected tocolumn chromatography (developing agent: petroleum ether (60-90° C.):ethyl acetate =1: 1), and following products can be obtained:

(1) 2-(1-benzyl-1H-indazole-3-oxyl)diethyl malonate (S2-1)

Yield: 39%

IR (KBr, cm⁻¹):2981 (m),1768 (s),1748 (s),1619 (m),1235 (s),744 (m)

EI-MS: 382 [M⁺]

¹HNMR(500 MHz, CDCl₃),δ(ppm):1.29˜1.33(t, 6H, CH₂CH₃), 4.29˜4.33(dd, 4H,CH₂CH₃),5.34(s, 2H, CH₂C₆H₅), 5.77(s, 1H, CH(COOH)₂), 7.08˜7.83(m, 9H,2Ar)

(2) 2-[1-(4-methyl-benzyl)-1H-indazole-3 -oxyl] diethyl malonate (S2-2)

Yield: 29%

IR(KBr, cm⁻¹): 2985(w),1764(s),1743(s),1617(m),1225(s),749(m)

EI-MS: 396 [M^(+])

¹HNMR(500 MHz, CDCl₃),δ(ppm): 1.28˜1.31(t, 6H, CH₂CH₃), 2.40(s, 3H,CH₂C₆H₄CH₃), 4.29˜4.33(dd, 4H, CH₂CH₃), 5.36(s, 2H, CH₂C₆H₄CH₃), 5.79(s,1H, CH(COOH)₂), 7.02˜7.80(m, 8H, 2Ar)

(3) 2-[1-(3-fluorine-benzyl)-1H-indazole-3-oxyl] diethyl malonate (S2-3)

Yield: 56%

IR(KBr, cm⁻¹): 2983(w),1767(s),1748(s),1618(m),1253(s),745(m)

EI-MS: 400 [M^(+])

¹HNMR(500 MHz, CDCl₃),δ(ppm): 1.28˜1.33(t, 6H, CH₂CH₃), 4.27˜4.34(dd,4H, CH₂CH₃),5.33(s, 2H, CH₂C₆H₄F), 5.76(s, 1H, CH(COOH)₂), 6.80˜7.83(m,8H, 2Ar)

(4) 2-[1-(3-chloro-benzyl)-1H-indazole-3-oxyl] diethyl malonate (S2-4)

Yield: 51%

IR(KBr, cm⁻¹): 2984(m),1735(s),1619(w),1247(s),747(m)

ESI-MS: 439 [M+Na]⁺

¹HNMR(500 MHz, CDCl₃),δ(ppm): 1.28˜1.33(t, 6H, CH₂CH₃), 4.31˜4.35(dd,4H, CH₂CH₃),5.29(s, 2H, CH₂C₆H₄Cl), 5.76(s, 1H, CH(COOH)₂), 7.02˜7.83(m,8H, 2Ar)

(5) 2-[1-(4-chloro-benzyl)-1H-indazole-3-oxyl] diethyl malonate (S2-5)

Yield: 18%

IR(KBr, cm⁻¹): 2982(w),1764(s),1740(s),1616(w),1222(s),746(m)

ESI-MS: 439 [M+Na]⁺

¹HNMR(500 MHz, CDCl₃),δ(ppm): 1.27˜1.34(t, 6H, CH₂CH₃), 4.28˜4.32(dd,4H, CH₂CH₃),5.29(s, 2H, CH₂C₆H₄Cl), 5.74(s, 1H, CH(COOH)₂), 7.04˜7.81(m,8H, 2Ar)

EXAMPLE 2

Preparation of 2-(1- Benzyl (or Substituent Benzyl)-1H-Indazole-3-Oxyl)Malonic Acids

The esters (2.7 mmol) obtained from the example 1 are added into 10 mlaqueous solution of 0.3 g potassium hydroxide (5.4 mmol) respectively,and the mixture is heated and subjected to reflux for 2h, subsequentlythe pH of the mixture is adjusted to 2 by 1M hydrochloric acid, whitesolids are precipitated, filtered and washed with small amount of water,and the following compounds can be obtained after drying:

(1) 2-(1-benzyl-1H-indazole-3-oxyl) malonic acid (H₂L¹)

Yield: 93%

Mp: 182-186° C. (decomposition)

IR(KBr, cm⁻¹):3033(m),2934(m),1741(s),1620(m),1257(s), 746(m), 711(m)

ESI-MS: 325 [M−H]⁻

¹HNMR(500 MHz, D₂O),δ(ppm): 5.30(s, 2H, CH₂C₆H₅), 5.36(s, 1H,CH(COOH)₂), 7.00˜7.73(m, 9H, 2Ar);

(2) 2-[1-(4-methyl-benzyl)-1H-indazole-3-oxyl)malonic acid (H₂L²)

Yield: 98%

Mp: 144-146° C.

IR(KBr, cm⁻¹):3023(m),2920(m),1755(s),1618(m),1255(s), 744(s), 727(m)

ESI-MS: 379 [M+K]⁺

¹HNMR(500 MHz, D₂O),δ(ppm): 2.16(s, 3H, CH₂C₆H₄CH₃), 5.29(s, 2H,CH₂C₆H₄CH₃), 5.46(s, 1H, CH(COOH)₂), 6.97-7.75(m, 8H, 2Ar);

(3) 2-[1-(3-fluorine-benzyl)-1H-indazole-3-oxyl]malonic acid (H₂L³)

Yield: 96%

Mp: 138-140° C.

IR(KBr, cm⁻¹):3065(m),2925(w),1732(s),1618(m),1254(s),743 (s)

ESI-MS: 383 [M+K]⁺, 367 [M+Na]⁺, 345 [M+H]⁺

¹HNMR(500 MHz, D₂O),δ(ppm): 5.41(s, 2H, CH₂C₆H₄F), 5.49(s, 1H,CH(COOH)₂), 6.82˜7.85(m, 8H, 2Ar);

(4) 2-[1-(3-chloro-benzyl)-1H-indazole-3-oxyl] malonic acid (H₂L⁴)

Yield: 98%

Mp: 137-139° C.

IR(KBr, cm⁻¹):3063(m),2940(m),1749(s), 1619(m),1247(s), 745(s), 725(m)

ESI-MS: 399 [M+K]⁺, 361 [M+H]⁺

¹HNMR(500 MHz, D₂O),δ(ppm): 5.40(s, 2H, CH₂C₆H₄Cl), 5.51(s, 1H,CH(COOH)₂), 7.04˜7.85(m, 8H, 2Ar);

(5) 2-[1-(4-chloro-benzyl)-1H-indazole-3-oxyl] malonic acid (H₂L⁵)

Yield: 88%

Mp: 114-116° C.

ESI-MS: 399 [M+K]⁺, 361[M+H]⁺

IR(KBr, cm⁻¹):3064(m),2921(m),1747(s), 1619(m),1251(s), 740(s)

¹HNMR(500 MHz, D₂O),δ(ppm): 5.32(s, 2H, CH₂C₆H₄Cl), 5.46(s, 1H,CH(COOH)₂), 7.01˜7.78(m, 8H, 2Ar)

EXAMPLE 3

Preparation of 2-(1-benzyl (or substituent benzyl)-1H-indazole-3-oxyl)malonates 2.0 mmol acids obtained from the example 2 are added into 5 mLwater respectively and agitated for 5 min, 5 mL aqueous solution of 4.0mmol alkali or 4.0 mmol amino acids is dropwise added, subsequently themixture is agitated at room temperature for 4h and concentrated underreduced pressure to remove water, and then oil-like substance isobtained, afterwards 20 mL dehydrated alcohol is added, the mixture isfiltered after solids are precipitated, the filter cake is washed with 2mL dehydrated alcohol and then subjected to vacuum drying, finally thefollowing products can be obtained:

(1) 2-(1-benzyl-1H-indazole-3-oxyl) malonate disodium (Y1)

Yield: 75%

IR(KBr, cm⁻¹):2983(w),1643(s),1334(m),741(m),723(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 5.21(s, 1H, CH(COO⁻)₂), 5.33(s, 2H,CH₂C₆H₅), 7.03˜7.79(m, 9H, 2Ar)

(2) 2-(1-benzyl-1H-indazole-3-oxyl) malonate dipotassium (Y2)

Yield: 86%

IR(KBr, cm⁻¹): 2978(w),1639(s),1328(m),740(m),721(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 5.23(s, 1H, CH(COO⁻)₂), 5.35(s, 2H,CH₂C₆H₅), 6.97˜7.78(m, 9H, 2Ar)

(3) 2-(1- benzyl-1H-indazole-3-oxyl) malonate diammonium (Y3)

Yield: 75%

IR(KBr, cm⁻¹):3197(s),1593(s),1326(s),1105(m),771(s),746(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 5.22(s, 1H, CH(COO⁻)₂), 5.34(s, 2H,CH₂C₆H₅), 7.03˜7.81(m, 9H, 2Ar)

(4) 2-(1-benzyl-1H-indazole-3-oxyl) malonate dihistidine (Y4)

Yield: 92%

IR(KBr, cm⁻¹):3128(m),3026(m),1618(s),1329(m),1259(w),744(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 3.10˜3.24(m, 4H, 2CH₂ of Histidine),3.89˜3.94(m, 2H, 2CHNH₂COOH of Histidine), 5.28(s, 1H, CH(COO⁻)₂),5.34(s, 2H, CH₂C₆H₅), 7.05˜7.80(m, 11H, 9H of 2Ar, and 2H of 2Imidazole), 8.27(s, 2H, 2H of 2 Imidazole)

(5) 2-(1- benzyl-1H-indazole-3-oxyl) malonate dilysine (Y5)

Yield: 86%

IR(KBr, cm⁻¹):3031(s),2941(s),1615(s),1325(m),741(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 1.30˜1.43(m, 4H, 2H₂NC₂H₄CH₂CH₂ of Lysine),1.60˜1.63(m, 4H, 2H₂NC₂H₄CH₂CH₂ of Lysine), 1.78˜1.82(m, 4H,2H₂NCH₂CH₂C₂H₄ of Lysine), 2.90˜2.93(m, 4H, 2H₂NCH₂CH₂C₂H₄ of Lysine),3.65(m, 2H, 2CH(NH₂)COOH of Lysine), 5.24(s, 1H, CH(COO⁻)₂), 5.36(s, 2H,CH₂C₆H₅), 7.09˜7.81(m, 9H, 2Ar)

(6) 2-[1-(4-methyl-benzyl)-1H-indazole-3-oxyl) malonate disodium (Y6)

Yield: 76%

IR(KBr, cm⁻¹):2983(m),1621(s),1336(s),1256(m),746(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 2.15(s,3H, C₆H₄CH₃), 5.22(s, 1H, CH(COO⁻)₂),5.27(s, 2H, CH₂C₆H₄CH₃), 6.97˜7.80(m, 8H, 2Ar)

(7) 2-[1-(4-methyl-benzyl)-1H-indazole-3-oxyl) malonate dipotassium (Y7)

Yield: 80%

IR(KBr, cm⁻¹):2986(m),1625(s),1338(m),1255(m),744(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 2.17(s,3H,C₆H₄CH₃), 5.25(s, 1H, CH(COO⁻)₂),5.30(s, 2H, CH₂C₆H₄CH₃), 6.99˜7.85(m, 8H, 2Ar)

(8) 2-[1-(4-methyl-benzyl)-1H-indazole-3-oxyl) malonate diammonium (Y8)

Yield: 70%

IR(KBr,cm⁻¹):3330(s),2983(m),1600(s),1331(m),746(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 2.13(s, 3H, C₆H₄CH₃), 5.21(s, 1H,CH(COO⁻)₂), 5.28(s, 2H, CH₂C₆H₄CH₃), 6.89˜7.80(m, 8H, 2Ar)

(9) 2-[1-(4-methyl-benzyl)-1H-indazole-3-oxyl) malonate dilysine (Y9)

Yield: 88%

IR(KBr, cm⁻¹):3432(s),2930(s),1619(s),1335(m),743(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 1.3˜1.38 (m, 4H, 2H₂NC₂H₄CH₂CH₂ of Lysine),1.54˜1.64(m, 4H, 2H₂NC₂H₄CH₂CH₂ of Lysine), 1.74˜1.81(m, 4H,2H₂NCH₂CH₂C₂H₄ of Lysine), 2.12(s, 3H, CH₂C₆H₄CH₃), 2.86˜2.91(m, 4H,2H₂NCH₂CH₂C₂H₄ of Lysine), 3.61˜3.65(m, 2H, 2CH(NH₂)COOH of Lysine),5.20(s, 2H, CH₂C₆H₅CH₃), 5.28(s, 1H, CH(COO⁻)₂), 6.92˜7.76(m, 8H, 2Ar)

(10) 2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxyl) malonate disodium (Y10)

Yield: 83%

IR(KBr, cm⁻¹):2927(w),1619(s),1337(m), 739(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 5.22(s, 1H, CH(COOH)₂), 5.34(s, 2H,CH₂C₆H₄F), 6.77˜7.80(m, 8H, 2Ar)

(11) 2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxyl) malonate dipotassium(Y11)

Yield: 80%

IR(KBr, cm⁻¹):2929(w),1605(s),1331(m), 741(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 5.22(s, 1H, CH(COOH)₂), 5.30(s, 2H,CH₂C₆H₄F), 6.75˜7.80(m, 8H, 2Ar)

(12) 2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxyl) malonate diammonium(Y12)

Yield: 75%

IR(KBr, cm⁻¹): 3340(s),2931(w),1641(s),1336(m), 743(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 5.35(s, 1H, CH(COOH)₂), 5.43(s, 2H,CH₂C₆H₄F), 6.77˜7.78(m, 8H, 2Ar)

(13) 2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxyl) malonate dihistidine(Y13)

Yield: 84%

IR(KBr, cm⁻¹): 3154(s),3012(m),1600(s),1331(m),1226(w),741(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 3.10˜3.13(d, 4H, 2CH₂ of Histidine),3.83˜3.87(m, 2H, 2CHNH₂COOH of Histidine), 5.22(s, 1H, CH(COO⁻)₂),5.26(s, 2H, CH₂C₆H₄F), 6.71˜7.73(m, 10H, 2Ar and 2H of 2 Imidazole ofHistidine), 8.33(s, 2H, 2 Imidazole of Histidine)

(14) 2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxyl) malonate dilysine (Y14)

Yield: 85%

IR(KBr, cm⁻¹): 3435(s),2931(s),1622(s),1328(m),744(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 1.32˜1.46(m, 4H, 2H₂NC₂H₄CH₂CH₂ of Lysine),1.55˜1.66(m, 4H, 2H₂NC₂H₄CH₂CH₂ of Lysine), 1.72˜1.79(m, 4H,2H₂NCH₂CH₂C₂H₄ of Lysine), 2.89˜2.93(m, 4H, 2H₂NCH₂CH₂C₂H₄ of Lysine),3.55˜3.59(m, 2H, 2CH(NH₂)COOH of Lysine), 5.25(s, 1H, CH(COO⁻)₂),5.35(s, 2H, CH₂C₆H₄F), 6.80˜7.87(m, 8H, 2Ar)

(15) 2-[1-(3-chloro-benzyl)-1H-indazole-3-oxyl] malonate disodium (Y15)

Yield: 80%

IR(KBr, cm⁻¹):2937(w),1619(s),1336(s),744(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 5.22(s, 1H, CH(COOH)₂), 5.26(s, 2H,CH₂C₆H₄Cl), 6.94˜7.80(m, 8H, 2Ar)

(16) 2-[1-(3- chloro-benzyl)-1H-indazole-3-oxyl] malonate dipotassium(Y16)

Yield: 78%

IR(KBr, cm⁻¹):2940(w),1605(s),1337(s),746(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 5.39(s, 1H, CH(COOH)₂), 5.43(s, 2H,CH₂C₆H₄Cl), 7.08˜7.95(m, 8H, 2Ar)

(17) 2-[1-(3 -chloro-benzyl)-1H-indazole-3-oxyl] malonate diammonium(Y17)

Yield: 71%

IR(KBr, cm⁻¹): 3250(s),2942(m),1641(s),1322(m),746(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 5.23(s, 1H, CH(COOH)₂), 5.33(s, 2H,CH₂C₆H₄Cl), 6.76˜7.81(m, 8H, 2Ar)

(18) 2-[1-(3 -chloro-benzyl)-1H-indazole-3 -oxyl] malonate dihistidine(Y18)

Yield: 82%

IR(KBr, cm⁻¹) :3132(s),2932(m),1640(s),1326(m),742(m)

¹HNMR(300 MHz,D2O),δ(ppm): 3.10 (m, 4H, 2CH₂ of Histidine), 3.81˜3.83(m,2H, 2CHNH₂COOH of Histidine), 5.16(s, 1H, CH(COO⁻)₂), 5.22(s, 2H,CH₂C₆H₄Cl), 6.86˜7.69(m, 10H, 8H of 2Ar and 2H of 2 Imidazole ofHistidine), 8.32(s, 2H, 2 Imidazole of Histidine)

(19) 2-[1-(3 -chloro-benzyl)-1H-indazole-3-oxyl] malonate dilysine (Y19)

Yield: 91%

IR(KBr, cm⁻¹):2934(m₂),1613(s),,1320(m),1105(m),741(m₄)

¹HNMR(500 MHz, D₂O),δ(ppm): 1.26˜1.34(m, 4H, 2H₂NC₂H₄CH₂CH₂ of Lysine),1.49˜1.56(m, 4H, 2H₂NC₂H₄CH₂CH₂ of Lysine), 1.72˜1.74(m, 4H,2H₂NCH₂CH₂C₂H₄ of Lysine), 2.81˜2.86(m, 4H, 2H₂NCH₂CH₂C₂H₄ of Lysine),3.52˜3.62(m, 2H, 2CH(NH₂)COOH of Lysine), 5.13(s, 2H, CH₂C₆H₄Cl),5.21(s, 1H, CH(COO⁻)₂), 6.88˜7.76(m, 8H, 2Ar)

(20) 2-[1-(4-chloro-benzyl)-1H-indazole-3-oxyl] malonate disodium (Y20)Yield: 84%

IR(KBr, cm⁻¹):2980(w),1642(s),1336(m),739(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 5.16(s, 2H, CH₂C₆H₄Cl), 5.22(s, 1H,CH(COOH)₂), 6.92˜7.76(m, 8H, 2Ar)

(21) 2-[1-(4-chloro-benzyl)-1H-indazole-3-oxyl] malonate dipotassium(Y21)

Yield: 82%

IR(KBr, cm⁻¹): 2982(w),1636(s),1325(m),741(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 5.18(s, 1H, CH(COOH)₂), 5.24(s, 2H,CH₂C₆H₄Cl), 6.99˜7.83(m, 8H, 2Ar)

(22) 2-[1-(4-chloro-benzyl)-1H-indazole-3-oxyl] malonate diammonium(Y22)

Yield: 78%

IR(KBr,cm⁻¹): 3232(s),2985(w),1612(s),1323(m),743(m)

¹HNMR(500 MHz,D₂O),δ(ppm): 5.24(s, 1H, CH(COOH)₂), 5.31(s, 2H,CH₂C₆H₄Cl), 7.08˜7.96(m, 8H, 2Ar)

(23) 2-[1-(4-chloro-benzyl)-1H-indazole-3-oxyl] malonate dihistidine(Y23)

Yield: 94%

IR(KBr, cm⁻¹):3162(m),2978(m),1638(s),1315(m),746(m)

¹HNMR(500 MHz, D₂O),δ(ppm): 3.00˜3.14 (m, 4H, 2CH₂ of Histidine),3.81˜3.85(m, 2H, 2CHNH₂COOH of Histidine), 5.19(s, 1H, CH(COO⁻)₂),5.25(s, 2H, CH₂C₆H₄Cl), 6.93˜7.71(m, 10H, 8H of 2Ar and 2H of 2Imidazole of Histidine), 812(s, 2H, 2 Imidazole of Histidine)

(24) 2-[1-(4-chloro-benzyl)-1H-indazole-3-oxyl] malonate dilysine (Y24)

Yield: 93%

IR(KBr, cm⁻¹):2930(m),1619(s),1316(m),1108(m),740(s)

¹HNMR(500 MHz, D₂O),δ(ppm): 1.24˜1.32(m, 4H, 2H₂NC₂H₄CH₂CH₂ of Lysine),1.48˜1.58(m, 4H, 2H₂NC₂H₄CH₂CH₂ of Lysine), 1.65˜1.67(m, 4H,2H₂NCH₂CH₂C₂H₄ of Lysine), 2.80˜2.85(m, 4H, 2H₂NCH₂CH₂C₂H₄ of Lysine),3.45˜3.53(m, 2H, 2CH(NH₂)COOH of Lysine), 5.19(s, 1H, CH(COO⁻)₂),5.21(s, 2H, CH₂C₆H₄Cl), 6.95˜7.75(m, 8H, 2Ar)

Activities of Typical Compounds

Some typical compounds are selected for the research on the capabilityof in vitro resisting oxidative damage to crystalline lens induced byH₂O₂. The experiments are illustrated as follows:

1. Culture of Eye Crystalline Lens

Kill rats and rabbits, take out their eyeballs and rinse with salinecontaining 500 u/mL penicillin, split the eyeball from the posterior toremove eyeball wall and vitreous body, strip off the suspensoryligament, take out the crystalline lens and wash with PBS solutioncontaining 500 u/mL penicillin and 0.5 mg/mL streptomycin for 5 minBefore experiment, check with anatomical lens to confirm that there isno opacification or other abnormal condition in the crystalline lens,then place the crystalline lens in the sterilized 12-well platecontaining 2 mL DMEM/high glucose (a product from Beijing Thermo FisherScientific Biochemical Product Co., Ltd.) sterile culture solution(which contains 100 u/mL penicillin and 0.1 mg/mL streptomycin), adddifferent compounds for screening and pre-culture in a incubator withtemperature of 37° C., humidity of 95% and 5% CO₂ for 1 h, subsequentlyadd 2 mL DEME culture solution containing H₂O₂ and FeCl₃ (namely, total4 mL culture solution in each well) (the total concentration of H₂O₂ ineach well is 2% for rabbits, and 5% for rats, and the concentrations ofFeCl₃ are all 0.02%), culture under the same conditions for 24 hours.The experiments are divided into several groups as below and carried outin batches, with each batch provided with negative control group andmodel group.

A. Negative control group: DMEM culture solution without H₂O₂.

B. Model group (oxidative damage group): DMEM culture solutioncontaining H₂O₂ and FeCl₃.

C. Vitamin C group: besides the ingredients of the model group, furthercontaining vitamin C (the final concentration is 1 mmol/L).

D. Bendazac lysine (BDL) group (BDL is provided by Zhejiang ShapuaisiPharmaceutical Co. Ltd., and its purity is more than 98.5%): besides theingredients of model group, further containing BDL (the finalconcentration is 0.5 mmol/L).

E. Sample group: besides the ingredients of model group, furthercontaining sample (the final concentration is 0.5 mmol/L).

2. Morphological Observations

After 24 h culture, observe the opacification degree of eye crystallinelens, take photograph under the background with black “+” in differentthickness on white ground below the 12-well plate, score and divide intothree different grades:

-   -   − indicates normal transparent lens;    -   + indicates mild opacity (the first “+” is slightly ambiguous        but is still clearly visible, the second “+” is clear); degree I    -   ++ indicates moderate opacity (the second “+” is slightly        ambiguous but is still clearly visible, the third “+” is clear);        degree II    -   +++indicates that the lens are totally opacified (the third “+”        f is not clearly visible); degree III 3. Results

The results for the capabilities of the sample compounds in resistingoxidative damage to in vitro eye lens of rabbits induced by H₂O₂ areshown in table 1; and the results for the capabilities of the samplecompounds in resisting oxidative damage to in vitro eye lens of ratsinduced by H₂O₂ are shown in table 2.

According to table 1, the compounds showing inhibitory effects onoxidative damage to in vitro eye lens of rabbits induced by H₂O₂ are Y4and Y10, and the inhibitory effects of Y1, Y2, Y5 and Y19 on oxidativedamage to in vitro eye lens of rabbits induced by H₂O₂ are notsignificant.

According to table 2, the compounds showing inhibitory effects onoxidative damage to in vitro eye lens of rats induced by H₂O₂ are Y1,Y4, Y5, Y14 and Y19, and the inhibitory effects of Y2 and Y10 onoxidative damage to in vitro eye lens of rats induced by H₂O₂ are notsignificant.

TABLE 1 the capabilities of the sample compounds in resisting oxidativedamage to in vitro eye lens of rabbits induced by H₂O₂ Percentages fordifferent degrees % Observation Results + >++ − + ++ +++ Total − Degree(I) Degrees(I, II) Comments A (the negative 61 61 100 control group) B(the model 26 31 22 79 33 67 group) C (the VC group) 13 16 2 31 42 58 D(the BDL 9 3 12 75 25 ✓ group) Y1 4 3 7 57 43 Y2 2 3 2 7 29 71 Y4 14 721 67 33 ✓ Y5 2 2 2 6 33 67 Y10 4 1 5 80 20 ✓ Y19 3 3 100

Evaluation criteria: according to large amount of experiments on themodel group, the ratio between the percentage of different degrees oflens opacification in the groups and the percentage of different degreesof lens opacification in the model group is utilized as the definition,wherein, for slight improvement (√), the percentage of ++ (degrees II,III) should be lower than 67%, and the percentage of + (degree I) shouldbe higher than 33%; for moderate improvement (√√), the percentage of++(degrees II, III) further decreases; and the percentage of 0 (−)appears; for significant improvement (√√√), the percentage of ++(degrees II, III) and + (degree I) disappears, and the lens are almosttransparent.

TABLE 2 the capabilities of the sample compounds in resisting oxidativedamage to in vitro eye lens of rats induced by H₂O₂ Percentages %Observation Results + Degree >++ Degrees − + ++ +++ Total − (I) (II,III) Comments A (the negative 84 9 12 13 118 71 8 21 control group) B(the model 15 18 47 36 116 13 15 72 group) C (the VC group) 7 8 3 18 3961 D (the BDL 30 22 3 55 54 46 ✓ group) Y1 4 1 5 80 20 ✓ Y2 3 3 6 100 Y418 3 21 86 14 ✓ Y5 1 4 1 6 17 83 Y10 47 28 3 78 60 40 ✓ Y14 10 8 18 5644 ✓ Y19 18 10 4 32 56 44 ✓

Evaluation criteria: according to large amount of experiments on themodel group, the ratio between the percentage of different degrees oflens turbidity in the groups and the percentage of different degrees oflens turbidity in the model group is utilized as the definition,wherein, for slight improvement (√), the percentage of ++ (degrees II,III) should be lower than 72%, and the percentage of + (degree I) shouldbe higher than 20%; for moderate improvement (√√) the percentage of++(degrees II, III) further decreases; and the percentage of 0 (−)appears; for significant improvement (√√√), the percentage of ++(degrees II, III) and +(degree I) disappears, and the lens are almosttransparent.

1. A group of compounds containing organic dicarboxylic acid group, thecompounds being abbreviated as H₂L, the general structural formulathereof is shown as below:

wherein, R stands for hydrogen atom, C₁₋₃ alkyl, C₁₋₃ alkoxyl or halogenatom.
 2. The dicarboxylate of the compounds containing organicdicarboxylic acid group according to claim 1, characterized in that, itis formed through the reaction of the dicarboxylic acid group in thestructural formula and monovalent cation M and is abbreviated as M₂L,wherein, M stands for Na⁺, K⁺ or NH₄ ⁺.
 3. The dicarboxyate of thecompounds containing organic dicarboxylic acid group according to claim1, characterized in that, it is formed through the reaction of thedicarboxylic acid group in the structural formula and amino acids and isabbreviated as A₂H₂L, wherein, A stands for amino acids, the said aminoacid is lysine or histidine, the said lysine is L-lysine, D-lysine, orthe raceme composed of L-lysine and D-lysine, the said histidine isL-histidine, D-histidine, or the raceme composed of L-histidine andD-histidine.
 4. The method for preparing the compound containing theorganic dicarboxylic acid group according claim 1, characterizedin that

reacts with XCH(CO₂R′)₂ under alkaline conditions to obtain

which is then hydrolyzed under alkaline conditions and subject toacidification to obtain

wherein, X stands for Cl, Br or I atom, R stands for hydrogen atom, C₁₋₃alkyl, C₁₋₃ alkoxyl or halogen atom, R′ stands for C₁₋₄ alkyl.
 5. Themethod according to claim 4, characterized by that 1.0 mole of

is subjected to reflux reaction with 1.2 mole of XCH(CO₂R′)₂ at thepresence of 2.5 moles of potassium carbonate in glycol dimethyl ethersolvent to obtain

which is subjected to reflux hydrolysis in potassium hydroxide (orsodium hydroxide) solution, and then acidified to pH of about 2 withdiluted hydrochloric acid, finally

is obtained, X stands for Cl, Br or I atom, R stands for hydrogen atom,C₁₋₃ alkyl, C₁₋₃ alkoxyl or halogen atom, R′ stands for C₁₋₄ alkyl.